Blood is the most popular biofluid used in proteomics to identify disease biomarkers. The potential of blood plasma or serum in diagnosis can be realized by the large amount of research and money spend on their study.
Interestingly, blood is a treasure trove of disease biomarkers. Every cell in the body leave a record of its physiological state in the form of waste or signal molecules in the products that it sheds to blood.Hence blood samples can reveal lot about physiology and pathological states of body. This review describes the various methods involved in the identification of low abundant protein biomarkers in blood.
Identification of low abundant protein biomarkers using blood as the starting sample
Blood is the most popular biofluid used in proteomics to identify disease biomarkers. The potential of blood plasma or serum in diagnosis can be realized by the large amount of research and money spend on their study.
Interestingly, blood is a treasure trove of disease biomarkers. Every cell in the body leave a record of its physiological state in the form of waste or signal molecules in the products that it sheds to blood.. Hence, a small sample of blood could reveal the ongoing physiological and pathological states of tissues in the body(Liotta et al., 2003).
Important considerations for using blood as a starting sample
The first important consideration to be taken into account is the difference between plasma and serum. Plasma is the liquid component of the blood in which blood cells are suspended whereas, serum is protein solution left after the bulk of fibrinogen has been removed by conversion into fibrin clot, together with platelets. In addition, varying amount of other proteins also gets removed with fibrin clot by specific or non-specific interactions. And hence, the protein concentration of serum is less than plasma (Lum and Gambina, 1974, Landenson et al.: cited by Lundblad, 2005). Furthermore, the process/storage containers, time of clot retraction, centrifugation speed and temperature of storage also influence serum quality. The time between venipuncture and freezing, process/storage containers, centrifugation speed and temperature of storage are critical variables for plasma. Even though, it is difficult to control all these variables, but a standard operating procedure for blood collection could ensure reproducibility of the results(Lundblad, 2005).
Another factor to be considered is the dynamic qualitative and quantitative range of proteins in the blood. It is speculated that the dynamic range of protein concentration would be at least 106 or greater(Lundblad, 2005). Both serum and plasma have high protein content. But 99% of the blood protein consist of 22 proteins, major one being albumin, transferin, immunoglobulin and complement factors(Veenstra et al., 2005) (Figure 1). This dynamic range of proteins exceeds the analytical capability of current proteomic methods, thereby making detection of low abundance protein fraction which contains most of the undiscovered biomarkers extremely challenging(Tirumalai et al., 2003). The heterogeneity of plasma and serum makes prefractionation to remove highly abundant proteins, essential in proteomic analysis(Lundblad, 2005).
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Figure 1: Pie-chart showing relative contribution of proteins within plasma(Tirumalai et al., 2003).
The various steps involved in detection of low abundance proteins in blood samples are summarized below:
Prefractionation
Prefractionation is done to reduce the dynamic range of protein concentration for detecting lower abundance proteins in blood samples. It is done prior to 2-DE and MS analysis. Some of the major prefractionation techniques are discussed below.
Electrophoresis based methods:
- Multicomponent electrolyzers (MCE) with isoelectric membranes: MCEs with isoelectric membranes is a preparative version of IPG. They are used to purify proteins in a liquid vein by capturing them in an isoelectric trap formed by two immobiline membranes having a pI range that include the pI of the protein species under study. When this apparatus was used for the analysis of human plasma with a pH range of 3-6, an increase in acidic proteins was seen when compared to the whole plasma(Herbert and Righetti, 2000).
Frequently Asked Questions
What are the key challenges in identifying low abundant protein biomarkers using blood as a starting sample?
The primary challenges include the difference between plasma and serum, the dynamic range of protein concentrations in blood, and the presence of highly abundant proteins that mask the detection of lower abundance biomarkers.
What is the difference between plasma and serum, and why is it important?
Plasma is the liquid component of blood with blood cells suspended in it, while serum is the protein solution left after fibrinogen has been removed. Serum has a lower protein concentration than plasma due to the removal of fibrinogen and other proteins during clot formation. This difference can impact proteomic analysis.
Why is the dynamic range of protein concentration a problem?
The dynamic range, speculated to be 106 or greater, exceeds the analytical capability of current proteomic methods. This makes detecting low abundance proteins, which often contain important biomarkers, extremely difficult.
What percentage of blood protein do highly abundant proteins constitute?
Approximately 99% of the blood protein consists of about 22 highly abundant proteins, including albumin, transferrin, immunoglobulin, and complement factors.
What is prefractionation, and why is it necessary?
Prefractionation is a process used to reduce the dynamic range of protein concentration in blood samples before proteomic analysis. It removes highly abundant proteins, allowing for better detection of lower abundance proteins.
What are some electrophoresis-based prefractionation methods mentioned in the text?
Two methods discussed are: - Multicomponent electrolyzers (MCE) with isoelectric membranes, which purify proteins in a liquid vein. - GradiflowTM, a preparative electrophoretic system that separates proteins based on size and pI.
How does GradiflowTM work?
GradiflowTM separates proteins based on their size and pI. The charge of the protein is controlled by the running buffer, and size separation is achieved using polyacrylamide membranes with specific pore sizes. This compartmentalizes abundant proteins, improving the visibility of less abundant ones.
- Quote paper
- Rahul Ramachandran (Author), 2010, Identification of low abundant protein biomarkers using blood as the starting sample, Munich, GRIN Verlag, https://www.grin.com/document/271078
